Original Article Formononetin Suppresses the Proliferation of Human Non-Small Cell Lung Cancer Through Induction of Cell Cycle Arrest and Apoptosis

Int J Clin Exp Pathol 2014;7(12):8453-8461 www.ijcep.com /ISSN:1936-2625/IJCEP0002029

Original Article Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis

Yi Yang, Yi Zhao, Xinghao Ai, Baijun Cheng, Shun Lu

Department of Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China. *Equal contributors. Received August 21, 2014; Accepted September 15, 2014; Epub December 1, 2014; Published December 15, 2014

Abstract: Formononetin is a novel herbal isoflavonoid isolated fromAstragalus membranaceus and possesses anti- tumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.

Keywords: Human non-small cell lung cancer, formononetin, proliferation, cell cycle, apoptosis

Introduction Recent studies have shown that Astragalus membranaceus can be used to alleviate the Lung cancer is the leading cause of cancer- side-effects of cytotoxic antineoplastic drugs related death worldwide, in which 80% of lung [6-8]. Formononetin is one of the major isofla- cancers are non-small cell lung cancer (NSCLC) vonoid constituents isolated from Astragalus with poor therapeutic efficacy when diagnosed membranaceus and demonstrates diverse [1, 2]. Though there has been significant devel- pharmacological benefits. As a phytoestrogen, opment in clinical treatment of NSCLC, its prog- it exhibits a metabolic effect by upregulating nosis is still unsatisfactory due to the low rate interleukin-4 production in activated T cells via of 5-year survival [3]. Fortunately, there are increased AP-1 DNA binding activity [11]. many extracts from traditional Chinese medi- Formononetin also possesses anti-inflammato- cines showing better therapies to human ry activity by inhibition of arachidonic acid NSCLC, leading us a new way to treat lung can- release in HT-29 human colon cancer cells [12]. cer in the future [4, 5]. Accumulating evidences demonstrated the anticancer activity of formononetin on breast Traditional Chinese herbs are significant sourc- cancer [13], prostate cancer [14] and cervical es of drugs that serve as potential therapeutic cancer [15]. However, the inhibitory effect of compounds for cancer treatment. Astragalus formononetin on human lung cancer cells has membranaceus (Radix Astragali) has a long never been investigated. Therefore, the present history of medicinal use in traditional Chinese study aimed to explore the anti-proliferative medicine as an immunomodulating agent to effects of formononetin on lung cancer cells, treat diarrhea, anorexia and fatigue [6-8]. and further elucidate the molecular mechanism Formononetin and proliferation of human non-small cell lung cancer

Figure 1. Inhibitory effects of formononetin on NSCLC cell proliferation. Lung cancer cells were treated with different concentrations of formononetin (50, 100, 150 and 200 μM) for 12 h, 24 h and 48 h. MTT assay was performed to determine the proliferation of A549 (A), NCI-H23 (B) and 16HBE-T cells (C).

underlying the anti-tumor property on human ed into 96-well plates at the density of 3 × 104 lung cancer. (cells/well) and left to adhere overnight. Cells were incubated with formononetin from 0~200 Materials and methods μM. Then 10 ml of 5 mg/ml MTT was added and incubated in dark at 37°C for 2 h. The Reagents absorbance was determined with the wave- length of 492 nm. Formononetin (purity > 99%) was purchased from Sigma (St. Louis, MO, USA)). Dulbecco’s Cell cycle analysis modified Eagle’s medium (DMEM) culture medium, fetal bovine serum (FBS), phosphate-buff- Cells were seeded at the density of 1.0 × 106 ered saline (PBS), penicillin-streptomycin (PS) cells/well in a 6-well plate for 24 h, and then and 0.25% (w/v) trypsin/1 mM EDTA were pur- treated with formononetin. After 24 h, cells chased from Gibco (Grand Island, NY, USA). were washed twice with PBS, detached with trypsin and harvested. For cell cycle analysis, Cell culture cells were harvested and collected by centrifu- The human NSCLC cell line A549, NCI-H23 and gation, followed by fixation in ice-cold 70% eth- an immortalized human bronchial epithelial cell anol at -20°C overnight. Then, cells were col- line 16HBE-T were purchased from the lected and stained with 100 μl PI staining solu- American Type Culture Collection (Rockville, tion for 30 min in the dark followed by cell cycle MD) and cultured in DMEM supplemented with analysis.

10% FBS in an atmosphere containing 5% CO2 at 37°C. Apoptosis detection

MTT assay Apoptosis cells were detected with annexin V-FITC/PI according to the protocol of Annexin Cell proliferation was determined by MTT assay. V-FITC cell Apoptosis Detection Kit (BD, USA). To be brief, A549 and NCI-H23 cells were seed- To be brief, A549 and NCI-H23 cells were seed-

8454 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer ed in a 6-well plate for 24 h and treated with Results different concentrations of formononetin. Cells were then harvested and washed twice with Anti-proliferative effect of formononetin on ice-cold PBS. Cells were then stained with NSCLC cells annexin V-FITC and propidium iodide (PI) for 60 min in dark at room temperature in binding buf- A549 and NCI-H23 cells were exposed to different concentrations of formononetin (50, 100, fer. The cell apoptosis in A549 and NCI-H23 150 and 200 μM) for 12 h, 24 h and 48 h. MTT cells were detected by flow cytometry (FACSC- assay was performed to determine the effects alibur, USA). of formononetin on NSCLC cells proliferation. We found that formononetin treatment signifi- Western blot analysis cantly inhibited the proliferation of A549 and A549 cells were treated with different concen- NCI-H23 cells in a time- and dose-dependent manner (Figure 1A and 1B). By contrast, for- trations of formononetin for 48 h. Proteins were mononetin had no obvious inhibitory effect on separated by sodium dodecyl sulfate polyacryl- 16HBE-T cells (Figure 1C). These data demon- amide gel electrophoresis (SDS-PAGE) and sub- strated the anti-proliferative role of formonone- sequently transferred to PVDF (Millipore, tin on NSCLC cells. Bedford, MA, USA) membrane. The blots were blocked with 5% non-fat milk at room tempera- Formononetin induced cell cycle arrest and ture for 1 h and incubated with the appropriate promoted apoptosis in NSCLC cells primary antibody including anti-p21, cyclin A, cyclin D1 (Cell Signaling, Beverly, MA); cleaved A549 and NCI-H23 cells were treated with dif- caspase-3, bax, bcl-2, p53, p-p53 Ser15/S20 ferent concentrations of formononetin (100, and β-actin (Santa Cruz Biotechnology, Santa 150 and 200 μM) for 24 h and cell cycle distri- Cruz, CA). Then, the blots were incubated with bution was analyzed by flow cytometry. peroxidase-conjugated secondary antibody. Compared with the control group, administra- Bands were detected using western blot chemi- tion of A549 cells with formononetin dose- luminescence reagent (Bio-Rad, Hercules, CA, dependently increased the proportions of cells in G1-phase, and decreased the number of USA). cells in S phase (Figure 2A and 2B). Similar Luciferase reporter assay results were also observed in NCI-H23 cells after treatment with formononetin (Figure 2C A549 cells were seeded into 24-well plates and and 2D). Moreover, the apoptosis rate of NSCLC cultured overnight. Then cells were transfected cells was analyzed using Annexin V and PI stain- with pGL3-p53 firefly luciferase reporter plas- ing. Compared with the control group, for- mid and pRL-SV40 (Renilla luciferase) plasmid mononetin treatment significantly promoted (Promega, USA) with Lipofectamine 2000 the apoptosis rates of A549 (Figure 3A) and NCI-H23 (Figure 3B) cells in a concentration- (Invitrogen, USA) according to the manufactur- dependent manner. Collectively, these results er's protocol. 24 h post-transfection, the cells indicated that formononetin induced G1-phase were treated with the indicated concentrations cell cycle arrest and promoted apoptosis in of formononetin for 24 h. Cell lysates were NSCLC cells. used for luciferase activity assay according to the Dual Luciferase Reporter Assay kit (Pro- Formononetin mediates proteins expression mega, USA) protocol. related to cell cycle arrest and apoptosis

Statistical analysis To further understand the molecular basis of formononetin-induced cell cycle arrest, we Each experiment was performed in triplicate, examined the expression levels of cell cycle- and repeated at least three times. For statisti- related proteins with western blot. A549 cells cal analysis, all the data were presented as were treated with different concentrations of means ± SD and treated for statistics analysis formononetin (100, 150 and 200 μM) for 48 h by SPSS program. Comparison between groups followed by western blot analysis (Figure 4A). was made using ANOVA and statistically We found that exposure to formononetin dose- significant difference was defined as P < 0.05. dependently increased the protein expression

8455 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer

Figure 2. Formononetin induced cell cycle arrest in NSCLC cells. Lung cancer cells were treated with formononetin at indicated concentrations (100, 150 and 200 μM) for 24 h. The cell cycle distribution of A549 (A and B) and NCI- H23 (C and D) was analyzed by flow cytometry. *P < 0.05; **P < 0.01.

Figure 3. Formononetin promoted apoptosis in NSCLC cells, Lung cancer cells were treated with formononetin at indicated concentrations (100, 150 and 200 μM) for 24 h. Flow cytometry was performed to measure the apoptosis rate of A549 (A) and NCI-H23 (B) cells. *P < 0.05; **P < 0.01. of p21 (Figure 4B). The expression levels of the 4D) were significantly decreased in a does- G1-phase cell cycle regulatory proteins includ- dependent manner after treatment with for- ing cyclin D1 (Figure 4C) and cyclin A (Figure mononetin for 48 h.

8456 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer

Figure 4. Effect of formononetin on the expression of cell cycle-related proteins. A549 cells were treated with formononetin at different concentrations (100, 150 and 200 μM) for 48 h. (A) Then protein expression was analyzed by western blot using antibodies against p21, cyclin D1 and cyclin A. β-actin was used as an internal control. The expression of p21 (B), cyclin D1 (C) and cyclin A (D) were measured by relative band intensities. *P < 0.05; **P < 0.01.

8457 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer

Figure 5. Effect of formononetin on the expression of apoptosis- related proteins. A549 cells were treated with formononetin at different concentrations (100, 150 and 200 μM) for 48 h. (A) Expression levels of apoptosis-related proteins were measured by using antibodies against Cleaved caspase 3, bax and bcl-2. β-actin was used as an internal control. Relative band intensities were used for quantification of Cleaved caspase 3 (B), bax (C) and bcl-2 (D). *P < 0.05; **P < 0.01.

Figure 6. Formononetin promoted the activity of p53. A549 cells were treated with formononetin at different concentrations (100, 150 and 200 μM) for 48 h. A and B. Western blot was performed to determine the levels of p53 and p53 Ser15/20 phosphorylation. β-actin was used as an internal control. C. A549 cells were treated with the indicated concentrations of formononetin for 24 h and underwent dual luciferase reporter assays. *P < 0.05; **P < 0.01.

Next we determined the expression of apopto- we detected the protein expression of p53 in sis-related proteins in A549 cells after treat- A549 cells to further explore the mechanism of ment with formononetin at indicated doses formononetin’s anti-cancer effect. Results (Figure 5A). Considering that caspase activa- showed that the total p53 expression exhibited tion often correlates with apoptosis, we detect- an increasing trend in response to formonone- ed the induction of cleavage of caspase-3 and tin treatment. We also demonstrated that the observed an increased cleavage of caspase-3 phosphorylation levels of p53 at Ser20 and in A549 cells in a dose-dependent manner Ser15 was significantly upregulated in a dose- (Figure 5B). In addition, we found that the pro- dependent manner following formononetin apoptotic protein bax was increased, whereas treatment at different concentrations (Figure the anti-apoptotic protein bcl-2 was decreased 6A and 6B). In addition, dual luciferase reporter in a dose-dependent fashion after formonone- assay indicated that the transactivation ability tin treatment for 48 h (Figure 5C and 5D). of p53 was significantly elevated following for- Taken together, these results indicated that formononetin treatment (Figure 6C). These results mononetin administration modulated the suggested that p53 participated in formonone- expression of critical cell cycle regulators and tin’s effects on apoptotic induction in NSCLC pro/anti-apoptotic signals, leading to cell cycle cells. arrest and apoptosis in NSCLC cells. Discussion p53 was pivotal in formononetin-induced cell apoptosis Formononetin, a major isoflavone constituent of Radix Astragali, has been proposed to be p53, as a tumor suppressor, has been reported potentially anticarcinogenic agent against sev- to be involved in cell apoptosis [16]. Therefore, eral kinds of cancers such as liver cancer and

8458 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer breast cancer [12, 17]. However, its effect on treatment with formononetin. Collectively, the- NSCLC cells and the underlying molecular se above results suggested that formonone- mechanism have never been explored. Our tin’s inhibitory effect on cell growth acted study for the first time demonstrated that for- through inducing cell cycle arrest and apo- mononetin inhibited the NSCLC cells prolifera- ptosis. tion through induction of cell cycle arrest and apoptosis. The p53 tumor suppressor plays a crucial role in multi-cellular organisms, where it regulates Firstly, we evaluated the anti-proliferative effect cell apoptosis and cell cycle [26, 27]. Thus, it is of formononetin on human lung cancer cells. a tempting strategy for cancer therapy to MTT assay revealed that formononetin exhibit- reverse downregulation of p53 in cancer cells. ed inhibitory effects on NSCLC cells lines Intriguingly, we found that formononetin- including A549 and NCI-H23. However, no obvi- induced apoptosis in A549 cells was mediated ous effect on normal human bronchial epitheli- by accumulation of p53 protein expression. As al cells was observed after treatment with for- an important transcriptional factor, the activity mononetin. Taken together, these results of p53 is mainly regulated by post-translational showed that formononetin inhibited the prolif- modifications including phosphorylation, acety- eration of lung cancer cells. lation and ubiquitination [28-30]. Previous studies have demonstrated that the phosphor- Cell cycle is a precise process controlled by cell ylation of p53 at Ser15 or Ser20 could increase cycle checkpoints which guarantee the fidelity p53 stability and promote cell apoptosis [31, of cell division. Cell cycle arrest could be trig- 32]. In our study, formononetin treatment gered by exogenous/endogenous stimulating upregulated the phosphorylation levels of p53 factors and resulted in breakdown of cell divi- at Ser20 and Ser15 in a dose-dependent man- sion, cell death and apoptosis [18-20]. Previous ner. Moreover, dual luciferase assay showed study found that formononetin treatment that the transactional ability of p53 was signifi- decreased cyclin D1 mRNA and protein expres- cantly enhanced after formononetin adminis- sion, thus inducing cell cycle arrest in human tration. These results suggested that p53 par- breast cancer cells [21]. In present study, we ticipated in formononetin’s effect on apoptotic found that treatment with formononetin for 24 induction in NSCLC cells. h induced cell cycle arrest in G1-phase. More importantly, we found that treatment of for- In conclusion, our study demonstrated that formononetin for 24 h dose-dependently incr- mononetin inhibited the proliferation of lung eased the protein expression of p21, and cancer cells through induction of cell cycle decreased the expression levels of cell cycle arrest and apoptosis. Thus, our study provides regulatory proteins such as cyclin A and cyclin a new mechanism to explain the chemopreven- D1. tive effect of formononetin on cancer and sug- gests that formononetin might be a new poten- Apoptosis is the process of programmed cell tial medicine that could be used for lung cancer death which is characterized by typical cellular therapy. and molecular features such as cell shrinkage, externalization of phosphatidylserine and con- Acknowledgements densation of chromatin [22, 23]. Multiple studies confirmed the therapeutic effect of for- This study was supported by Science & mononetin on cancer treatment through induc- Technology Fund Planning Project of Shanghai tion of cell apoptosis and critical pro-apoptotic Chest Hospital (2014YZDC10600). proteins expression [24, 25]. We demonstrated Disclosure of conflict of interest that formononetin exposure for 24 h substan- tially increased cell apoptosis in a dose-depen- None. dent manner. On the molecular level, formononetin administration promoted the cleav- Address correspondence to: Dr. Shun Lu, Depart- age of caspase-3 and expression of the pro- ment of Oncology, Shanghai Chest Hospital, Shang- apoptotic protein bax. Meanwhile, the anti- hai Jiao Tong University, Shanghai 200030, China. apoptotic protein bcl-2 was significantly Tel: +86-21-22200000; E-mail: shun_lu@hotmail. decreased in a does-dependent manner after com

8459 Int J Clin Exp Pathol 2014;7(12):8453-8461 Formononetin and proliferation of human non-small cell lung cancer

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